The vertebrate immune system comprises an intricate system of cells, secreted factors, and responses for protecting an organism from pathogenic infection by microbes, viruses, toxins, and other pathogens and irritants. Certain molecules, however, comprise epitopes which do not induce an effective immune response in a vertebrate because of their small size and/or because they are endogenously synthesized within the vertebrate and are therefore not perceived as “foreign” by the vertebrate's immune system. Methods for producing antibodies against certain peptides which are normally non-immunogenic, such as hormones, are desirable because immunoregulation of the activity of such peptides within the organism can thereby be achieved.
Hormone peptides have been combined with various carrier peptides in fusion proteins to elicit an effective immune response against the hormone when an organism is vaccinated with the fusion protein. The carrier portion causes the organism's immune system to recognize and generate antibodies against the hormone peptide which it would not otherwise generate.
U.S. Pat. No. 5,403,586 to Russell-Jones et al., for example, relates to fusion proteins which comprise an analog of gonadotropin releasing hormone (GnRH), also known as luteinizing hormone releasing hormone (LHRH), and a TraTp analog, wherein the presence of the TraTp analog in the fusion protein helps trigger the production of anti-GnRH antibodies. TraTp is an outer membrane lipoprotein produced by certain strains of E.coli, as described in U.S. Pat. No. 5,403,586, above.
U.S. Pat. No. 5,422,110 to Potter et al. relates to carrier systems that include chimeric proteins which comprise a leukotoxin polypeptide fused to a selected antigen. The leukotoxin functions to increase the immunogenicity of the antigen. Selected antigens that are disclosed therein include GnRH, somatostatin (SRIF), and rotavirus viral protein 4 (VP4).
WO 90/02187 relates to fusion proteins which comprise an antigenic, hydrophilic portion, such as Hepatitis B surface antigen (HBsAg), and a peptide, such as GnRH, which alone is not substantially antigenic
GnRh is a decapeptide endogenously produced, mainly in the hypothalamus. It is highly conserved among vertebrate species. In mammals, the GnRH gene encodes the decapeptide glu-his-trp-ser-tyr-gly-leu-arg-pro-gly (SEQ ID NO: 13) with subsequent post-translational modification of the N and C termini to pyroglutamic acid and glycinamide, respectively. GnRH has been shown to play a critical role in the regulation of reproductive functions in all major vertebrates by regulating the production and release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland. Because FSH and LH play a role in spermatogenesis and ovulation, as well as steroidogenesis, vaccines that result in the production of antibodies against GnRH lead to the suppression of reproductive function (fertility) in both males and females, and should also control secondary sexual characteristics such as gender-related behavior. In males, LH regulates steroidogenesis in Leydig cells. Thus, active immunization of males against GnRH leads to testicular atrophy and a decrease in testosterone production and testicular function, (Ladd, A. et al., 1994, Biol. Reprod. 51:1076-1083; Ladd A., 1993, Am. J. Reprod. Immunol. 29:189-194). A GnRH vaccine has been approved by the United States Food and Drug Administration as an investigational new drug for the treatment of prostate cancer (Ladd A., 1993, above). The development of a GnRH immuno-contraceptive is a useful alternative to surgical sterilization in animals, and has the added advantage of being reversible, since spermatogeneis and fertility can return to normal by simply allowing anti-GnRH titers to decline (Ladd, A. et al., 1989, J. Reprod. Immunol. 15:85-101). However, since GnRH is a small self peptide and has a short half-life (WO 90/02187, Mar. 8, 1990), it is only weakly immunogenic, even when injected with a powerful adjuvant. For example, a significant proportion of animals are not able to mount an effective antibody response against GnRH when administered in Freund's complete adjuvant. In order to generate a significant antibody response, GnRH must therefore be conjugated, chemically or recombinantly, to a carrier protein.
None of the aforementioned references, however, teach or suggest using a carrier which triggers an immunoinhibiting response against itself.